Acanthamoeba T4 genotype associated with keratitis infections in Tunisia

Acanthamoeba keratitis (AK) is a sight-threatening infection. We report five cases of AK diagnosed from 2005 to 2009 in the Laboratory of Parasitology-Mycology at Habib Bourguiba Sfax Hospital, Tunisia. All were associated with improper care of contact lenses (rinsing of contact lenses with tap water and inappropriate cleaning) and lens storage. The patients displayed different clinical presentations: corneal inflammation, corneal ulceration, and corneal abscess. The diagnosis was made after direct examination, culture, and polymerase chain reaction amplification with specific primers. The genotype classification was based on the highly variable DF3 region in the 18S rRNA gene. This is the first study characterizing Acanthamoeba genotype in Tunisia and North Africa. All Acanthamoeba isolates were associated to the T4 genotype. Three different DF3 sequence types were related to AK infections T4/10, T4/15, and T4/16.     

Evaluation of Three Different Methods to Establish Animal Models of Acanthamoeba Keratitis

Purpose

To produce animal models of Acanthamoeba keratitis and to evaluate the advantages and adaptation range of each of the three methods employed.

Materials and Methods

Mice and Wistar rats in three groups of 15 rats and 15 mice each were used to establish the models. Right corneas in group A were scratched and challenged with Acanthamoeba. Those in group B were scratched and covered with contact lenses incubated with Acanthamoeba. Those in group C received an intrastromal injection of Acanthamoeba. Five rats and 5 mice in each group were used for histopathological investigations and the other 10 in each group were used for clinical evaluation. The models were evaluated by slit lamp examination, microscopic examination and culture of corneal scrapings, HE staining of corneal sections, and pathological scoring of the infections.

Results

Four rats and 6 mice in group A, 7 rats and 8 mice in group B, and 10 rats and 10 mice in group C developed typical Acanthamoeba keratitis.

Conclusion

Corneal scratching alone has the lowest infection rate, while scratching and then covering with contaminated contact lenses has a moderate rate of infection and most closely mimics what happens in most human infections. Intrastromal injection of Acanthamoeba gives a much higher infection rate and more severe Acanthamoeba keratitis.     

<Emphasis Type="Italic">Acanthamoeba</Emphasis> keratitis due to <Emphasis Type="Italic">Acanthamoeba</Emphasis> genotype T4 in a non-contact-lens wearer in Turkey

An otherwise healthy 22-year-old man presented with Acanthamoeba keratitis (AK) in the right eye. He was not a contact lens wearer and had no history of corneal trauma. The Acanthamoeba strain isolated from a corneal scraping was identified as morphological group II and genotype T4. Three more Acanthamoeba strains isolated from sites of possible human contact with acanthamoebae in the same geographical region, including a lens storage case, tap water and soil, were subjected to morphological and molecular biological identification. Whereas the strain from tap water also exhibited genotype T4, the two other isolates were identified as morphological group I and genotype T9. To the best of our knowledge, this is the first study identifying an AK-causing Acanthamoeba strain in Turkey and the first isolation of genotype T9 in this country.The authors declare that all experiments performed comply with the current laws of Turkey and Austria.     

First report of an Acanthamoeba genotype T13 isolate as etiological agent of a keratitis in humans

Several strains of free-living amoebae (FLA) belonging to the genus Acanthamoeba are able to cause a painful sight-threatening disease of the cornea designated as Acanthamoeba keratitis (AK). In this case report, a 22-year-old woman, wearer of soft contact lenses, was treated after the initial examination, and follow-up laboratory results led to the diagnosis of Acanthamoeba keratitis. The patient recovered under the targeted therapy, demonstrating that the acanthamoebae were the etiological agents of the keratitis in this case. The acanthamoebae belonged morphologically to group II. Genotyping of the causative Acanthamoeba strain based on sequences of the PCR amplimer ASA.S1 amplified from 18S ribosomal DNA by using the genus-specific primers JDP1 and JDP2 followed. The phylogenetic comparison of ASA.S1 confirmed that the isolated Acanthamoeba strain is closely related to genotype T13 supported by pairwise sequence identities of 97.1–98.0 % and bootstrap support of 980 replicates with reference sequences of genotype T13. These results regarding the Acanthamoeba keratitis-causing isolate KaBo expands the number of known pathogenic genotypes to 12. To our knowledge, this is the first report of a T13 Acanthamoeba genotype being associated with keratitis in humans.     

Resistance of Acanthamoeba Cysts to Disinfection in Multiple Contact Lens Solutions

Acanthamoebae are free-living amoebae found in the environment, including soil, freshwater, brackish water, seawater, hot tubs, and Jacuzzis. Acanthamoeba species can cause keratitis, a painful vision-threatening infection of the cornea, and fatal granulomatous encephalitis in humans. More than 20 species of Acanthamoeba belonging to morphological groups I, II, and III distributed in 15 genotypes have been described. Among these, Acanthamoeba castellanii, A. polyphaga, and A. hatchetti are frequently identified as causing Acanthamoeba keratitis (AK). Improper contact lens care and contact with nonsterile water while wearing contact lenses are known risk factors for AK. During a recent multistate outbreak, AK was found to be associated with the use of Advanced Medical Optics Complete MoisturePlus multipurpose contact lens solution, which was hypothesized to have had insufficient anti-Acanthamoeba activity. As part of the investigation of that outbreak, we compared the efficacies of 11 different contact lens solutions against cysts of A. castellanii, A. polyphaga, and A. hatchetti (the isolates of all species were genotype T4), which were isolated in 2007 from specimens obtained during the outbreak investigation. The data, generated with A. castellanii, A. polyphaga, and A. hatchetti cysts, suggest that the two contact lens solutions containing hydrogen peroxide were the only solutions that showed any disinfection ability, with 0% and 66% growth, respectively, being detected with A. castellanii and 0% and 33% growth, respectively, being detected with A. polyphaga. There was no statistically significant difference in disinfection efficacy between the 11 solutions for A. hatchetti.Acanthamoebae, which are free-living amoebae, occur worldwide in soil and water. It has been isolated from ponds, lakes, brackish water. and seawater; filters of heating, ventilating, and air-conditioning units; medical equipment, such as gastric wash tubing, dental irrigation units, contact lenses, and contact lens solutions; as well as vegetables, cell cultures, and even human and animal tissues (7, 23, 39). It has also been isolated from toxic waste dumpsites with high levels of pesticides, herbicides, pharmaceuticals, heavy metals, and polychlorinated biphenyls (35). Acanthamoeba species have two stages in their life cycle: a vegetative or trophozoite stage that reproduces by binary fission and that feeds voraciously on the bacteria and detritus present in the environment and a nondividing, cyst stage that is resistant to environmental stress. Acanthamoeba amoebae cause different types of human disease, including central nervous system infections (granulomatous amebic encephalitis, cutaneous infections) Acanthamoeba dermatitis, and ocular infections (Acanthamoeba keratitis [AK]). Granulomatous amebic encephalitis and cutaneous infections principally occur in immunocompromised individuals, including patients with human immunodeficiency virus infection or AIDS (17, 23, 37, 43). In contrast, AK principally occurs in immunocompetent individuals.AK is a painful vision-threatening infection, which, if it is not treated promptly, may lead to ulceration of the cornea, a loss of visual acuity, and, eventually, blindness (7, 15, 16). AK is associated with trauma to the cornea and with contact lens wear as a result of poor lens care and hygiene. When introduced into the eye by a contaminated contact lens, Acanthamoeba amoebae may adhere to the corneal surface and subsequently infiltrate the stoma and cause tissue damage (10). Both Acanthamoeba cysts and trophozoites can be isolated by culture from corneal scrapings or biopsy specimens and from contact lens paraphernalia (23, 43). Confocal microscopy has been used as an aid for the diagnosis of AK (29). Molecular techniques such as real-time PCR assays have been developed for the identification of Acanthamoeba species (32, 33). Sequencing analysis of the 18S rRNA gene has been used to identify as many as 15 genotypes of Acanthamoeba, of which the T4 genotype appears to be the most common in the environment and in patients with AK (2, 23).The first documented case of AK in the United States occurred in 1973 in a south Texas rancher following trauma to his right eye (15, 40, 42). Both trophozoite and cyst stages of Acanthamoeba polyphaga were demonstrated in corneal sections. Between October 1985 and August 1986, Stehr-Green et al. (41) conducted a case-control study to investigate an outbreak of AK in the United States. The majority of case patients wore contact lenses, and illness was most commonly associated with the use of homemade saline solutions and lens care practices, such as the disinfection of the lenses less frequently than recommended and swimming while wearing contact lenses (8, 41). Contact lens use is now considered an important risk factor for AK in the United States. AK cases have continued to be diagnosed since the 1986 outbreak, but because AK is not a reportable disease in the United States, the actual number of cases occurring each year is not known.A recent study indicated a dramatic increase in the number of AK cases in the Chicago, IL, area (16). An investigation conducted by the Centers for Disease Control and Prevention (CDC) revealed that this increase in the number of AK cases was occurring nationwide, starting in 2004 and continuing through 2007 (7). A subsequent investigation identified the use of Advanced Medical Optics (AMO) Complete MoisturePlus multipurpose contact lens solution as the primary risk factor, leading to an international recall of this product by the manufacturer (7, 16). We therefore decided to examine this and other frequently used major contact lens solutions for their efficacies against Acanthamoeba species isolated from clinical samples collected during the 2007 AK outbreak investigation.     

<Emphasis Type="Italic">Acanthamoeba</Emphasis> T4 and T15 genotypes associated with keratitis infections in Italy

Thus far there is little data available concerning Acanthamoeba associated amoebic keratitis (AK) from Italy. In order to understand the incidence of Acanthamoeba in patients with ocular infections and to characterize the isolates at the molecular level, ocular specimens and contact lenses or lens case solutions from 140 patients were analysed by culture and by an 18S rRNA (Rns) gene-based PCR method. Nineteen (13.6%) patients showed Acanthamoeba culture positive samples. Eleven out of the 14 genetically characterized isolates were assigned to the T4 genotype. Three isolates, two of them from patients with keratitis responding to specific anti-Acanthamoeba therapy, were identified as belonging to the T15 genotype. This finding represents the first association between the T15 genotype and human amoebic keratitis. PCR amplification of the 18S ribosomal DNA proved to be a sensitive method, potentially able to detect Acanthamoeba without the need of long culture incubation, and thus considerably useful for clinical applications.     

An extraordinary endocytobiont in Acanthamoeba sp. isolated from a patient with keratitis

In the present article, the detection and the development of a parasitic endocytobiont within host amoebae (Acanthamoeba sp.) recently isolated from the contact lens and the inflamed eye of a patient with keratitis is presented. An otherwise healthy 55-year-old female patient presented with keratitis in her inflamed left eye. She was a contact lens wearer and had no history of a corneal trauma. Acanthamoebae as well as other smaller free-living amoebae could be detected from the fluid of the contact lens storage cases by culture methods. A successful therapy could be provided consequently. Two of these Acanthamoeba strains showed intracellular aggregating organisms. Within 2 to 3 days, the host amoebae ruptured, and numerous microorganisms were released. We succeeded in detecting the mechanism of infection and intrusion of this organisms by using light and electron microscopy. Infection with this endocytobiont is a suitable model for studying the host–parasite relations while the parasites use their hosts as so-called Trojan horses (see Barker, Lambert, Brown, Infect Immun 61:3503–3510, 1992).     

Clinical and histologic evaluations of experimental <Emphasis Type="Italic">Acanthamoeba</Emphasis> keratitis

Amoebic keratitis, a sight-threatening, progressive corneal disease, is commonly caused by ubiquitous, pathogenic, free-living Acanthamoeba spp., which are widely distributed in the environment. We investigated clinical findings and histology of Acanthamoeba keratitis in a rat cornea model. Experimental Acanthamoeba keratitis was induced in Wistar rats by intrastromal inoculation of Acanthamoeba castellanii trophozoites. The clinic features of Acanthamoeba keratitis by day 70 are observed. All rats inoculated with Acanthamoeba developed keratitis. Histologically, the eyes displayed blood vessels, edema, and amoebae in stroma. A mixed cellular response, including neutrophils, mononuclear cells, and spindle-shaped cells, was seen. In conclusion, progressive, suppurative Acanthamoeba keratitis can be induced in the rat cornea model. This rat cornea model assists researchers who study the pathogenesis of Acanthamoeba keratitis and devise treatment for this difficult condition.     

Rapid and sensitive diagnosis of Acanthamoeba keratitis by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of Acanthamoeba. The sensitivity of the LAMP assay was tested using different copies of positive DNA. The specificity of the assay was tested using DNA extracted from Acanthamoeba, Pseudomonas aeruginosa, Candida albicans, herpes simplex virus-1 and human corneal epithelial cells. Its effectiveness was evaluated and compared with culture, corneal smear examination and real-time PCR in corneal samples from mice with Acanthamoeba keratitis. We also tested three corneal samples from patients with suspected Acanthamoeba or fungal infection using LAMP. Loop-mediated isothermal amplification was confirmed to be very sensitive, with the lowest detection limit being ten copies/tube of Acanthamoeba DNA. The LAMP primers only amplified Acanthamoeba DNA. During the development of Acanthamoeba keratitis in mice, almost all of the positive rates of LAMP at each time post-infection were higher than those of culture or corneal smear examination. The total positive rate of LAMP was significantly higher than those of culture and corneal smear examination (p <0.05), whereas the sensitivities of LAMP and real-time PCR were comparable. However, the trends of positive change in these different test methods were generally similar. Of the three clinical corneal specimens, two with suspected Acanthamoeba keratitis tested positive for Acanthamoeba using LAMP along with culture or corneal smear examination, whereas the other suspected fungal keratitis tested negative. The LAMP assay is a simple, rapid, highly specific and sensitive method for the diagnosis of keratitis caused by Acanthamoeba.     

A Rabbit Model of Acanthamoeba Keratitis That Better Reflects the Natural Human Infection

Acanthamoeba species are ubiquitous, free‐living protozoa that can invade the cornea and result in Acanthamoeba keratitis (AK), a painful progressive sight‐threatening corneal disease. Disease progression in current animal models is too rapid to mimic AK in humans accurately. This study provides a novel method for establishing AK in rabbits and compared it with the conventional method with regard to pathogenesis and immune response in humans. The New Zealand white rabbits were randomly divided into two experimental groups (Groups A and B). Rabbits in the Group A (n = 14) received intrastromal injections of 1 × 10⁴/100 µL Acanthamoeba healyi trophozoites (conventional AK model). The Group B animals (n = 14) received microinjections of 1 × 10⁴/10 µL A. healyi trophozoites between the corneal epithelium and Bowman's layer, anterior to the corneal stroma (novel AK model). In addition, two rabbits were left untreated as normal controls. AK in the treated rabbits was evaluated clinically, histopathologically, and immunologically for 35 days. AK was successfully established in both the conventional and novel model groups. Compared with the Group A, AK in the Group B displayed an efficient immune response with less severe pathology. Moreover, the self‐limiting but chronic nature of the infection in the Group B was strikingly similar to that of AK in humans. The novel animal model for AK described here more closely simulates the pathogenesis and immune response of Acanthamoeba corneal infection in humans than the animal models currently in use. Anat Rec, 298:1509–1517, 2015. © 2015 Wiley Periodicals, Inc.     

Laboratory Diagnosis of Amoebic Keratitis: Comparison of Four Diagnostic Methods for Different Types of Clinical Specimens

Amoebic keratitis causes significant ocular morbidity in contact lens wearers. Current diagnostic methods for amoebic keratitis are insensitive and labor-intensive and have poor turnaround time. We evaluated four laboratory methods for detection of acanthamoebae in clinical specimens. Deidentified, delinked consecutive specimens from patients with suspected amoebic keratitis were assayed for acanthamoebae by direct smear analysis, culture, and PCR using two different primer sets specific for Acanthamoeba ribosomal DNA. The consensus reference standard was considered fulfilled when the results for any two of the four tests were positive, and the outcome measures were sensitivity and specificity. Of 107 specimens assayed over an 18-month period, 20 were positive for acanthamoebae. The sensitivity and specificity of each assay were as follows, respectively: for smear analysis, 55% (95% confidence interval [CI], 33.2 to 76.8%) and 100%; for culture, 73.7% (95% CI, 54.4 to 93.0%) and 100%; for PCR using Nelson primers, 90% (95% CI, 76.9 to 100%) and 90.8% (95% CI, 84.7 to 96.9%); and for PCR using JDP primers, 65% (95% CI, 44.1 to 85.9%) and 100%. Nelson primer PCR demonstrated a single-organism level of analytic sensitivity. The performance characteristics of the assays varied by specimen type, with contact lenses and casings showing the highest rates of detectable acanthamoebae and the highest diagnostic sensitivities for direct smear analysis, culture, and JDP primer PCR, though these results are based on small numbers and should be interpreted cautiously. These findings have important implications for clinicians collecting diagnostic specimens and for diagnostic laboratories, especially in outbreak situations.Amoebic keratitis (AK) is a potentially blinding ocular infection caused by an Acanthamoeba sp. free-living protozoan parasite that is found ubiquitously throughout the environment worldwide (3). The overwhelming majority of cases of AK occur in immunocompetent contact lens wearers (14), and outbreaks have been linked to contact lens solutions contaminated with acanthamoebae or to those that fail to effectively decontaminate lenses. A recent outbreak in the United States affecting 138 people led to the recall of contact lens solutions and products by both the FDA and Health Canada and has resulted in over 150 lawsuits against the manufacturer (2, 6, 7). Plaintiffs in the lawsuits have been left with impaired vision and, in several cases, have required corneal transplants (7). Although contaminated contact lens solutions or solutions that facilitate growth are usually implicated in large outbreaks of AK, isolated cases occur in individuals who have corneal trauma or who disinfect contact lenses with tap water or other home-based preparations. Swimming and showering while wearing contact lenses are also risk factors for AK. Annual incidences of AK vary by country and are believed to be on the order of 2 to 20 cases per million contact lens wearers, accounting for 10% of the North American population (8, 12, 16, 17, 19, 21).Clinically, AK can be easily mistaken for herpes simplex virus infection or fungal keratitis, and secondary bacterial infection is common, thus complicating diagnosis (10). Delayed diagnosis has repeatedly been associated with poor visual outcome and more-severe clinical progression (4, 5). Standard laboratory diagnostic procedures include microscopic examination of Giemsa-, periodic acid Schiff-, hematoxylin-and- eosin-, or acridine orange-stained corneal scrapings or contact lens fluids and culture of these specimens on nonnutrient agar overlaid with Escherichia coli or Klebsiella pneumoniae, and all of these procedures are limited by poor sensitivity, the requirement for technical expertise, and, in the case of culture, long turnaround time (4, 5). Due to their excellent sensitivity, molecular methods, including PCR, are increasingly being used to detect acanthamoebae in corneal specimens (9, 10, 18, 20).As suggested by the recent outbreak and legal/medicolegal sequelae, strategies which improve upon current diagnostic methods for AK are needed (4). We herein sought to evaluate the performance of PCR for detection of acanthamoebae in clinical specimens from patients suspected of having AK in comparison to traditional methods, such as direct microscopic examination and culture.     

Development of an Immunochromatographic Assay Kit Using Fluorescent Silica Nanoparticles for Rapid Diagnosis of Acanthamoeba Keratitis

We developed an immunochromatographic assay kit that uses fluorescent silica nanoparticles bound to anti-Acanthamoeba antibodies (fluorescent immunochromatographic assay [FICGA]) and evaluated its efficacy for the detection of Acanthamoeba and diagnosis of Acanthamoeba keratitis (AK). The sensitivity of the FICGA kit was evaluated using samples of Acanthamoeba trophozoites and cysts diluted to various concentrations. A conventional immunochromatographic assay kit with latex labels (LICGA) was also evaluated to determine its sensitivity in detecting Acanthamoeba trophozoites. To check for cross-reactivity, the FICGA was performed by using samples of other common causative pathogens of infectious keratitis, such as Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans. Corneal scrapings from patients with suspected AK were tested with the FICGA kit to detect the presence of Acanthamoeba, and the results were compared with those of real-time PCR. The FICGA kit detected organisms at concentrations as low as 5 trophozoites or 40 cysts per sample. There were no cross-reactivities with other pathogens. The FICGA was approximately 20 times more sensitive than the LICGA for the detection of Acanthamoeba trophozoites. The FICGA kit yielded positive results for all 10 patients, which corresponded well with the real-time PCR results. The FICGA kit demonstrated high sensitivity for the detection of Acanthamoeba and may be useful for the diagnosis of AK.     

Acanthamoeba keratitis

Acanthamoeba keratitis is being recognized with increasing frequency worldwide, and appears to have a predilection for wearers of soft contact lenses. Unless recognized and treated early, this infection may result in permanent blindness or loss of the affected eye. The clinical laboratory, in consultation with the ophthalmologist, plays a pivotal role in assisting with the diagnosis. If possible, both direct examination (including permanently stained preparations) and culture of corneal specimens should be undertaken. These techniques are easily performed in most clinical laboratories and should be attempted to maximize chances of recovery. Unfamiliarity with these organisms may be the limiting factor for laboratorians attempting to detect their presence.     

Evaluation of Loop-mediated Isothermal Amplification Assay for Rapid Diagnosis of Acanthamoeba Keratitis

Background: The clinical features of Acanthamoeba keratitis (AK) are non-specific and closely resemble bacterial, viral and fungal keratitis. Materials and Methods: We compared loop-mediated isothermal amplification (LAMP) with microscopy, non-nutrient agar (NNA) culture and polymerase chain reaction (PCR) in clinical suspects of AK. Results: Of 52 clinical samples (42 AK suspects and 10 proven bacterial, viral or fungal keratitis), 3 were positive by direct microscopy (sensitivity 60%, confidence interval [CI]: 17%–92.7%), and 5 by NNA culture, 18S rDNA PCR and LAMP (sensitivity 100%, CI: 46.3%–100%). The limit of detection of Acanthamoeba DNA was 1 pg/μl by both LAMP and PCR. Conclusion: PCR and LAMP assays targeting 18S rDNA gene were found particularly suitable for a rapid and accurate diagnosis of AK. LAMP assay takes 2–3 h lesser than PCR, and thus offers a rapid, highly sensitive and specific, simple and affordable diagnostic modality for patients suspected of AK, especially in resource limited settings     

Contact lens-related polymicrobial keratitis: Acanthamoeba spp. genotype T4 and Candida albicans

A 31-year-old female daily user of contact lenses sought medical attention, reporting blurred vision and irritation of the left eye. Slit-lamp examination revealed hyperemia and an irregular corneal epithelium surface, and empirical treatment was started. A corneal scrape was obtained and examined for the presence of fungi, bacteria, and Acanthamoeba spp. The results of the microbial culture revealed growth of Acanthamoeba spp. and Candida albicans. The Acanthamoeba isolate was characterized by cyst morphology as belonging to group II according to Pussard and Pons. Sequencing of the diagnostic fragment 3 (DF3) region located on the 18S ribosomal DNA identified the isolate as genotype T4. The patient was treated with chlorhexidine 0.02% and polyhexamethylene biguanide (PHMB) 0.02% drops for 5 months until the infection resolved. Lately, rare cases of polymicrobial keratitis associated with Acanthamoeba and Candida albicans have been reported. Cases of co-infection are more difficult to treat, since the specific treatment depends on precise identification of the agents involved.

     

18S ribosomal DNA typing and tracking of Acanthamoeba species isolates from corneal scrape specimens, contact lenses, lens cases, and home water supplies of Acanthamoeba keratitis patients in Hong Kong

We examined partial 18S ribosomal DNA (Rns) sequences of Acanthamoeba isolates cultured in a study of microbial keratitis in Hong Kong. Sequence differences were sufficient to distinguish closely related strains and were used to examine links between strains obtained from corneal scrape specimens, contact lenses, lens cases, lens case solutions, and home water-supply faucets of patients with Acanthamoeba. We also looked for evidence of mixed infections. Identification of Acanthamoeba Rns genotypes was based on sequences of approximately 113 bp within the genus-specific amplicon ASA.S1. This permitted genotype identification by using nonaxenic cultures. Of 13 specimens obtained from corneal scrapes, contact lenses, lens cases, or lens case solutions, 12 were Rns genotype T4 and the remaining one was Rns genotype T3. The sequences of corneal scrape specimens of two patients also were the same as those obtained from their contact lenses or lens case specimens. A possible triple-strain infection was indicated by three different T4 sequences in cultures from one patient's lenses. Although faucet water used by patients to clean their lenses is a possible source of infections, specimens isolated from the faucets at two Acanthamoeba keratitis patients' homes differed from their corneal scrape or lens specimens. The overall results demonstrate the potential of this Rns region for tracking Acanthamoeba keratitis strains in infections and for distinguishing single-strain and closely related multiple-strain infections even when other microorganisms might be present with the cultured specimens. They also confirm the predominance of Rns genotype T4 strains in Acanthamoeba keratitis infections.     

Molecular Identification of T4 and T5 Genotypes in Isolates from Acanthamoeba Keratitis Patients

Acanthamoeba keratitis (AK) is a rare but sight-threatening ocular infection. Outbreaks have been associated with contaminated water and contact lens wear. The epidemiology and pathology may be associated with unique genotypes. We determined the Rns genotype for 37 clinical isolates from 23 patients presenting at the University of Miami Bascom Palmer Eye Institute with confirmed AK infections in 2006 to 2008. The genus-specific ASA.S1 amplicon allowed for rapid genotyping of the nonaxenic cultures. Of the 37 isolates, 36 were of the T4 genotype. Within this group, 13 unique diagnostic fragment 3 sequences were identified, 3 of which were not in GenBank. The 37th isolate was a T5, the first in the United States and second worldwide to be found in AK. For five patients with isolates from the cornea and contact lens/case, identical sequences within each patient cluster were observed, confirming the link between contact lens contamination and AK infection. Genotyping is an important tool in the epidemiological study of AK. In this study, it allowed for the detection of new strains and provided an etiological link between source and infection. Additionally, it can allow for accurate categorizing of physiological differences, such as strain virulence, between isolates and clades.The genus Acanthamoeba is comprised of a group of free-living amoebae that are responsible for causing Acanthamoeba keratitis (AK), a rare but sight-threatening corneal infection. In recent years, the number of AK cases has been on the increase, especially among wearers of contact lenses, who make up 85 to 90% of the AK cases (8, 24, 31, 35). Diagnosis of AK is problematic due to clinical features which are similar to those of herpetic, bacterial, and fungal infections. For example, the stromal ring infiltrate associated with AK is only observed ∼6% of the time in early cases and ∼16% of the time in late cases (2, 3, 4, 14). AK can be the primary infection or be present as a suprainfection in combination with other infectious organisms, like bacteria or fungi, thereby complicating diagnosis and treatment. The encystment capability of Acanthamoeba species also confounds treatment due to the recalcitrant nature of the cyst to most treatment options allowing reemergence of amoebae after treatment cessation.Acanthamoeba genotyping is a useful tool for studying taxonomic and epidemiological relationships and thereby allowing correlations between the infectious isolates and disease phenotypes, such as virulence factors, drug susceptibility, and/or species-clinical outcome correlations, to be explored. The gene targeted most often in Acanthamoeba genotyping is the nuclear small-subunit rRNA gene (Rns), and utilizing a 5% sequence dissimilarity cutoff point, 15 or more genotype clades, designated T1, T2, T3, etc., have been identified (12, 13, 15, 17, 29). Isolates from six of the genotypic clades (T3, T4, T5, T6, T11 and T15) are confirmed to be causative agents of AK (10, 13, 19, 21, 28, 29, 34, 36). The most prevalent Acanthamoeba genotype in both clinical and environmental samples is the T4 genotype (6, 7). Within the genotype clades, multiple species designations can be observed. This is primarily due to the traditional classification method''s reliance on changeable morphological characteristics, such as cyst morphology, creating inconsistent species identification (25, 32). Therefore, it was proposed that each genotypic clade be equated with a single species (29). For example, all isolates in the T4 clade could be reclassified as Acanthamoeba castellanii since the T4 genotype includes the type strain for that species.In this study, 37 isolates from corneal scrapes, contact lenses, and lens cases of 23 patients presenting with AK at the Anne Bates Leach Eye Hospital, Bascom Palmer Eye Institute, University of Miami, from 2006 to 2008 were examined to assess the Rns genotypes responsible for the infections. Acanthamoeba species can be rapidly genotyped by targeting a highly variable region designated diagnostic fragment 3 (DF3) within the genus-specific Rns ASA.S1 amplicon (5, 26); therefore, this region was chosen for analysis. The genotypes identified in this study were also compared to strains identified in other studies in order to examine the prevalence of the DF3 sequence types within genotype clades (5, 37, 38).     

Early diagnosis of amoebic keratitis due to a mixed infection with <Emphasis Type="Italic">Acanthamoeba</Emphasis> and <Emphasis Type="Italic">Hartmannella</Emphasis>

A mixed keratitis due to Acanthamoeba and Hartmannella species is reported. The patient was a soft contact lens wearer. Early diagnosis was achieved by polymerase chain reaction and culture. The pathogenic potential of the isolated amoebae was proven using cytotoxicity assays. The reported case underlines the difficulties in identifying a corneal amoebic infection. In our case, the early diagnosis of a mixed infection allowed a proper antiamoebic treatment in an early stage of infection. This may have been the reason of a successful outcome after therapy.     

Morphological, physiological and molecular biological characterisation of isolates from first cases of Acanthamoeba keratitis in Slovakia

Acanthamoeba keratitis (AK) is a relatively rare disease worldwide. Over the past 10 years, five cases of AK were reported in Slovakia. Four preserved Slovak strains and one strain from the Czech Republic isolated from corneal scrapes of patients with AK are characterised in this study. Genotype identification of isolates is based on sequences of the PCR amplimer GTSA.B1 amplified from 18S ribosomal DNA. A strain isolated from the first patient in 1999 was classified as a rare sequence type T15. This is just the second report in which genotype T15 has been associated with AK. The other three Slovak strains were identified as belonging to the most common genotype T4. The only strain originating from the Czech Republic was classified as sporadically appearing sequence type T3. All isolates were also studied for their temperature tolerance and growth characteristics. The cythopatic effect was tested in vitro on Vero cell cultures.     

Acanthamoeba DNA can be directly amplified from corneal scrapings

This study evaluated the performance of direct amplification of Acanthamoeba-DNA bypassing DNA extraction in the diagnosis of Acanthamoeba keratitis in clinically suspected cases in comparison to direct microscopic examination and in vitro culture. Corneal scrapings were collected from 110 patients who were clinically suspected to have Acanthamoeba keratitis, 63 contact lens wearers (CLW), and 47 non-contact lens wearers (NCLW). Taken samples were subjected to direct microscopic examination, cultivation onto the non-nutrient agar plate surface seeded with Escherichia coli, and PCR amplification. The diagnostic performance of these methods was statistically compared. The results showed that Acanthamoeba infection was detected in 21 (19.1 %) of clinically suspected cases (110); 17 (81 %) of them were CLW and the remaining 4 (19 %) positive cases were NCLW. Regarding the used diagnostic methods, it was found that direct amplification of Acanthamoeba DNA bypassing nucleic acid extraction was superior to microscopy and culture in which 21 cases (19.1 %) were positive for Acanthamoeba by PCR compared to 19 positive cases by culture (17.3 %) and one case (0.9 %) by direct smear. The difference in detection rates between culture and direct smear was highly statistically significant (P?=?0.001). On the other hand, there was no significant difference in detection rates between culture and PCR (P?=?0.86). On using culture as the gold standard, PCR showed three false-positive samples that were negative by culture and one false-negative sample that was positive by culture. At the same time, direct smear showed 18 false-negative samples. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of PCR were 94.7, 96.7, 85.7, 98.9, and 96.4, respectively, while those of direct smear were 5.3, 100, 100, 83.5, and 83.6, respectively. In conclusion, direct amplification of Acanthamoeba-DNA bypassing DNA extraction is a reliable, specific, sensitive method in the diagnosis of Acanthamoeba keratitis in clinically suspected cases. It should set up in ophthalmological centers as an easy diagnostic tool.